The present invention relates to the field of chemical and biochemical reactions. More specifically, the present invention relates to the use of photogenerated reagents (PGR) for use in parallel synthesis and assay of a plurality of organic and bio-organic molecules on a substrate surface in accordance with a predetermined spatial distribution pattern. Methods and apparatus of the present invention are useful for preparing and assaying very-large-scale arrays of DNA and RNA oligonucleotides, peptides, oligosacchrides, phospholipids and other biopolymers and biological samples on a substrate surface.
Development of modern medicine, agriculture, and materials imposes enormous demands on technological and methodological progress to accelerate sample screening in chemical and biological analysis. Development of parallel processes on a micro-scale is critical to the progress. Many advances have been made in this area using parallel synthesis, robotic spotting, inkjet printing, and microfluidics (Marshall et al., Nature Biotechnol. 16, 27-31 (1998)). Continued efforts are sought for more reliable, flexible, faster, and inexpensive technologies.
For high-throughput screening applications, a promising approach is the use of molecular microarray (MMA) chips, specifically biochips containing high-density arrays of biopolymers immobilized on solid surfaces. These biochips are becoming powerful tools for exploring molecular genetic and sequence information (Marshall et al., Nature Biotechnol. 16, 27-31 (1998) and Ramsay, Nature Biotechnol. 16, 40-44 (1998)). Target molecules have been hybridized to DNA oligonucleotides and cDNA probes on biochips for determining nucleotide sequences, probing multiplex-interactions of nucleic acids, identifying gene mutations, monitoring gene expression, and detecting pathogens (Schena, et al., Science 270, 467460 (1995); Lockhart et al., Nature Biotechnol. 14, 1675-1680; Weiler, Nucleic Acids Res. 25, 2792-2799 (1997); de Saizieu et al., Nature Biotechnol. 16, 45-48 (1998); Drmanc et al., Nature Biotechnol. 16, 54-58 (1998)). The continued development of biochip technology will have a significant impact on the fields of biology, medicine, and clinical diagnosis.
Light-directed on-chip parallel synthesis has been used in the fabrication of very-large-scale oligonucleotide arrays with up to one million sequences on a single chip. Two major methods have been disclosed: synthesis using photolabile-group protected monomers (Pirrung et al., U.S. Pat. No. 5,143,854 (1992); Fodor et al., U.S. Pat. No. 5,424,186 (1995)) and synthesis using chemical amplification chemistry (Beecher et al., PCT Publication No. WO 98/20967 (1997)). Both methods involve repetitive steps of deprotection, monomer coupling, oxidation, and capping. Photomasks are used to achieve selective light exposure in predetermined areas of a solid substrate surface, on which oligonucleotide arrays are synthesized.
For the synthesis process involving photolabile-protecting groups, the photolabile-protecting groups are cleaved from the reactant, i.e., the 5′-O of the growing oligonucleotide molecules in illuminated surface areas while in non-illuminated surface areas the protecting groups on oligonucleotide molecules are not affected. The substrate surface is subsequently contacted with a solution containing monomers having an unprotected first reactive center and a second reactive center protected by a photolabile-protecting group. In the illuminated surface areas, monomers couple via the unprotected first reactive center with the deprotected oligonucleotide molecules. However, in the non-illuminated surface areas oligonucleotides remain protected with the photolabile-protecting groups and, therefore, no coupling reaction takes place. The resulting oligonucleotide molecules after the coupling are protected by photolabile protecting groups on the second reactive center of the monomer. Therefore, one can continue the above photo-activated chain propagation reaction until all desired oligonucleotides are synthesized.
There are significant drawbacks in the method involving photolabile-protecting groups: (a) the chemistry used is non-conventional and the availability of building blocks is limited (only DNA oligonucleotides are now routinely made); (b) the method is not applicable to the synthesis of other types of organic molecules due to the unavailability of the photolabile protected building blocks; (c) the method suffers from low sequence fidelity due to inherent low efficiency of the photoreaction used and requirement of 100% deprotection efficiency.
The method of using chemical amplification chemistry has its limitations as well: (a) The method requires application of a polymer/photoresist layer and is not suitable for routine solution reactions since there is no measure provided for separating sites of reaction on a solid surface; (b) in certain circumstances, destructive chemical conditions required for pre- and post-heating and stripping the polymer/photoresist layer cause the decomposition of oligonucleotides on solid surfaces; (c) the entire process is labor intensive and difficult to automate due to the requirement for many cycles (up to 80 cycles if 20-mers are synthesized) of photoresist coating, heating, alignment, fight exposure and stripping; (d) the method is not applicable to a broad range of biochemical reactions or biological samples to which chemical amplification reagents are applied since embedding of biological samples in such a polymer/photoresist layer may be prohibitive.
Additional limitations are linked to the use of photomasks in the above two methods: (a) Setup for making a new chip is expensive and time consuming due to a large number of photomasks that have to be made; (b) photolithography equipment is expensive and complicated, and thus, can not be accessed by many interested users; (c) photolithography processes have to be conducted in an expensive cleanroom facility and require trained technical personnel. These limitations undermine the applications of oligonucleotide chips and the development of the various MMA-chips.
Recently a new method for producing biopolymers on biochips and microarrays has been developed. U.S. Pat. No. 6,426,184 describes an apparatus and methods for synthesizing and assays of arrays of biopolymers utilizing PGRs. These PGRs are useful in the synthesis and assays of arrays of biopolymers by virtue of the fact that the precursors of these reagents can be used in conventional synthesis reactions to produce biopolymers with high yield or that the precursors of these reagents can be used in conventional reaction conditions to induce changes in reaction conditions. The production of the PGRs is accomplished by the irradiation of these PGR precursors, which undergo photolytic reaction upon irradiation to produce product or intermediate that can be utilized in synthesis and other chemical reactions.
While not intended for solution reactions, there are ample examples of PGR-P compounds, which are used as polymerization initiators and as reagents used in chemical amplification reactions of photoresists (a polymer matrix). These processes are fundamental to microelectronic fabrication of semiconductor industries. Examples of PGR-P compounds are photogenerated acid precursors (PGA-P) that yield H+ in the form of carboxylic acids, phosphate acids, sulfate acids, and hydrohalogen acids. PGA-P may also be Lewis acids, forming complexes, such as MmXn (m and n are number of atoms). Examples of PGR-P compounds also include photogenerated base precursors (PGB-P) that yield a base, such as an amine, a hydroxide or the like, upon irradiation. References for such compounds may be found in Süs et al., Liebigs Ann. Chem. 556, 65-84 (1944); Hisashi Sugiyama et al., U.S. Pat. No. 5,158,855 (1997); Cameron et al., J. Am. Chem. Soc. 113, 4303-4313 (1991); Frechet, Pure & Appl. Chem. 64, 1239-1248 (1992); Patchornik et al., J. Am. Chem. Soc. 92, 6333-6335 (1970). PGA-P compounds have been widely used for many years in printing and microelectronics industries as a component in photoresist formulations (Willson, in “Introduction to microlithography”, Thompson et al. Eds., Am. Chem. Soc.: Washington D.C., (1994)). A specific example of a PGA-P is triarylsulfonium hexafluoroantiimonate derivatives (Dektar et al. J. Org. Chem. 53, 1835-1837 (1988); Welsh et al., J. Org. Chem. 57, 4179-4184 (1992); DeVoe et al. Advances in Photochemistry 17, 313-355 (1992)). This compound-belongs to a family of onium salts, which undergo photodecompositions, either directly or sensitized, to form free radical species and finally produce diarylsulfides and H+.
The PGA chemical amplification reaction has recently been modified and applied to an imaging process (acid amplified imaging or AAI, Marshall et al. Science 297, 1516 (2002)). In these solid-phase reactions, sensitizer dyes, super sensitizer, iodonium photo-acid generator, and amplifier reagents are present in thin layers with or without the presence of a polymer matrix, such as polystyrene (the binder). It is believed that the AAI reagents are not dissolved in the polymer. Light irradiation activates sensitizers which react with iodonium photo acid generator to produce primary H+. The light activated reaction is accelerated under after heating the thin layers of the AAI reagents to high temperature (140° C.). The system can be stabilized after the AAI reactions by light bleaching the sensitizers, reducing the iodonium salts using hydroquinone reducing agents (fixer), and base neutralizing the acid generated.
The PGA compounds have been shown in solution reactions to be effective for parallel synthesis of microarrays of oligonucleotides and peptides (Gao et al. Nucleic Acids Res. 29, 4744-4750 (2001); Pellois et al., Nature Biotechnol. 20, 922-926 (2002)).